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Fig. 1. Confocal FRAP measurements of the lateral translational selfdiffusion coef-
ficient of 2500 kDa aggrecan and 870 kDa hyaluronan, as a function of concentration.
These data show aggrecan forms a more dynamic network than hyaluronan, because
of its more compact branched structure.
chains. Comparison of the self diffusion of aggrecan and hyaluronan shows a
contrast between the extended stiffened random coil behavior of hyaluronan
with the more compact conformation of aggrecan (
Fig. 1
). Investigations of
the permeability of aggrecan networks to FITC-dextran solutes demonstrate
the effects of size-selective molecular sieving (
Fig. 2
) with the mobility of a
2000 kDa probe being reduced much more than a 167 kDa probe as the
concentration of aggrecan rises.
Experimentally, confocal-FRAP involves viewing a solution of a
fluorescently labeled molecule with a confocal microscope, locally creating a
bleached area in the solution, followed by observing the subsequent redistribu-
tion of fluorescence. The bleaching can be either continuous or, as described
below, it can be achieved rapidly by brief high-power laser illumination. In
simple solutions of monodisperse macromolecules, the measurement of the
recovery of fluorescence yields a long-time translational lateral self diffusion
coefficient
(17)
. This describes the movement of the macromolecule through a
matrix of like macromolecules. Importantly, the technique measures self diffu-
sion coefficients under equilibrium conditions, which in the absence of shear,
or flow, favors any weak intermolecular interactions that may stabilize the net-
work. The technique is also well suited to measure the mobility of a labeled
probe molecule within a macromolecular network in solution. This measures
the lateral diffusion coefficient of the tracer from which the effective pore
dimensions of the matrix can be calculated. It thus enables the effects of com-
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